Method of using cyclodextrin

ABSTRACT

Described are methods for using cyclodextrin to treat, inhibit, prevent, ameliorate, or cure diabetes or conditions relating to diabetes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 claimingpriority to international application, PCT/US2013/036484, filed Apr. 12,2013, which claims priority to or the benefit of U.S. provisional patentapplication, Ser. No. 61/624,087, filed Apr. 13, 2012, which areincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

This invention generally relates to a method for lowering plasmamembrane and cellular cholesterol for the prevention, treatment, cure,or reversal of obesity, metabolic syndrome, diabetes, or a complicationrelating thereto, by administering to a patient in need thereof, acompound, such as a cyclodextrin or derivative thereof, to lower plasmamembrane and cellular cholesterol and/or lipid.

Obesity, metabolic syndrome, pre-diabetes and diabetes represent aworldwide epidemic with a major health care cost, as all these prevalentmedical conditions are major risk factors for cardiovascular morbidityand mortality.

It is well established that elevated plasma cholesterol levels and, inparticular, low-density lipoprotein (LDL) cholesterol levels, play animportant role in the development of coronary heart disease, stroke,peripheral vascular disease, kidney disease, atherosclerosis andhypertension. Clinical trials have demonstrated that decreasingcholesterol concentrations in the plasma has a major impact oncardiovascular morbidity and mortality. Therefore, therapeutic controlof systemic hypercholesterolemia is critically important in the clinicalmanagement and treatment of obesity, metabolic syndrome, pre-diabetesand diabetes.

While correction of systemic hypercholesterolemia in vascular relateddisorders has been extensively studied, strategies to reduce localcholesterol accumulation at the plasma membrane and cellular level intarget organs of obesity, metabolic syndrome, prediabetes and diabeteshave not been studied.

Accumulation of cholesterol in patients with obesity, metabolic syndromeor diabetes has been described in several organs such as pancreas,muscle, liver, blood vessels, kidneys, although the functionalconsequences of accumulation of local cholesterol in these organsremains to be established.

In both clinical and experimental renal disease, for example, renalaccumulation of cholesterol correlates with the development ofglomerulosclerosis, and kidneys from diabetic rats are characterized bycholesterol accumulation. Excessive accumulation of cholesterol may bedeleterious to cell function though several mechanisms including amodulation of cellular actin cytoskeleton, a modulation of the responseof podocytes to several circulating factors (insulin, IGFs, VEGF, anygrowth factor, apolipoproteins, adipokines, endocrine hormones), amodulation of locally produced inflammatory cytokines, chemokines andtheir receptors, integrins, a modulation of the immune response (such asthe one mediated through TLRs and co-stimulatory molecules asB7-1-CD80), or a modulation of pro- and anti-apoptotic cell deathpathways.

Furthermore we have recently demonstrated an important role of asphingolipid related enzymes (acid sphingomyelinase-likephosphodiesterase 3b, SMPDL3b) as modulators of podocyte function, whichare kidney cells heavily affected in diabetes. Similarly,glycosphingolipids accumulate in the kidney of diabetic rats. However,no data are available yet showing a beneficial effect of plasma membraneor cellular cholesterol removal in proteinuric or other renal-relateddiseases such as diabetic kidney diseases.

Therefore, what is needed is an intervention method that can modulatethe accumulation of plasma membrane and cellular cholesterol observed indisease. Such method may then be useful in a preventive and therapeuticstrategy for treating, inhibiting or ameliorating such conditions asdiabetes, prediabetes, metabolic syndrome, obesity, or symptoms thereof.

Cyclodextrins (CD) are cyclic oligosaccharides that contain 6 or moreD-(+) glucopyranose units that are attached by α, β, or γ-(1,4)glucosidic bonds. It has been shown that cyclodextrins are able to formcomplexes with a variety of hydrophobic molecules due to their uniquestructure. Cyclodextrin derivatives are extensively used in researchlabs, for example to remove cholesterol from cultured cells and they arewell known in the pharmaceutical industry for their ability tosolubilize drugs.

Underivatized cyclodextrins are used throughout the food industry tomake cholesterol-free products, such as fat-free butter, eggs and milkproducts. Hydroxypropyl-beta-cyclodextrin (HPβCD) is recognized as aGRAS (Generally Recognized As Safe) material for use in food products inAsian and European countries and is being considered for similarcertification in the United States. Millions of people worldwide areexposed to small amounts of cyclodextrin compounds every day in food,cosmetics and household products. CD derivatives are commerciallyavailable from CTD Holdings Inc. (http://cyclodex.com/). Pharmacologicalstudies have suggested that the cholesterol depleting capacities of CDderivatives is approximately 20 times superior to the cholesteroldepleting capacities of statins.

In addition to this common exposure to cyclodextrins, the potentialsafety and efficacy for CD use in therapies has also been recognized.For example, in April 2009, the US Food and Drug Administration (FDA)approved an Investigational New Drug (IND) protocol that allowed twinswith a rare brain-destroying cholesterol disease called Niemann Pickdisorder to undergo weekly intravenous infusions of HPRCD into theirbloodstreams. However, subsequent research discovered thatHydroxypropyl-beta-cyclodextrin does not cross from the bloodstream intothe brain. And on Sep. 23, 2010, the FDA granted clearance of an INDapplication to introduce Trappsol® Cyclo™ (HPβCD) into the brains of sixyear old identical twin girls dying from Niemann Pick Type C (NPC).

A summary of the IND submission for this indication and route ofadministration is publicly available at the address(http://addiandcassi.com/wordpress/wp-content/uploads/2009/09/Hydroxy-Propyl-Beta-Cyclodextrin-HPBCD-Summary.pdf).

What has not been previously described are uses of CD and CD derivativesfor reducing cellular cholesterol or lipids of obesity, metabolicsyndrome, pre-diabetes and diabetes or any related complications.

Advantageously, it has now been discovered that cyclodextrin compoundsdo not act by affecting the cholesterol synthetic pathway (as do thestatins) but primarily by increasing the efflux mechanisms leading tocholesterol accumulation.

This discovery allows for cyclodextrin compounds to be more broadlyutilized for the prevention and the cure of obesity, metabolic syndrome,pre-diabetes and diabetes or any related complications. Moroever,cyclodextrin derivatives or any cellular cholesterol-lowering agentbelonging to a class of compounds other than statins (such as chromiumpicolinate, liver X receptor agonists) can also be used in a method asdescribed herein.

One novel strategy to modulate cellular cholesterol include themodulation of sphingolipid related enzymes such as SMPDL3B(sphingomyelin phosphodiesterase, acid-like 3B), which we havedemonstrated to modulate cellular cholesterol content and cell function

SUMMARY OF THE INVENTION

This invention relates to the method of administering to a patient inneed thereof, a compound, such as a cyclodextrin or a cyclodextrinderivative, to lower plasma membrane or cellular cholesterol/lipid for atime and at a dose sufficient to prevent, treat, cure, or reverseobesity, metabolic syndrome, pre-diabetes, diabetes, diabetic kidneydisease or conditions or symptoms relating thereto.

Generally, the invention concerns a method for reducing lipid content ina cell or plasma membrane of a cell in a patient suffering from acondition selected from diabetes (type 1), diabetes (type 2),prediabetes, obesity, metabolic syndrome, diabetic nephropathy, diabetickidney disease, diabetic neuropathy, diabetic retinopathy, diabetesrelated microvascular complication, diabetes related macrovascularcomplications, atherosclerosis, peripheral vascular disease, coronaryartery diseases, congestive heart failure, cardiac hypertrophy,myocardial infarction, endothelial dysfunction and hypertension, stroke,cerebrovascular accident, myocardial infarction, heart attack,cardiovascular accidents, fatty liver, steatohepatitis, NASH, andinsulin resistance, diabetic kidney disease, obesity, and metabolicsyndrome, said method comprising administering to the patient aneffective amount of a compound which reduces cellular lipid content.

More specifically, a method of the invention comprises inhibition of acellular influx mechanism or increases a cellular efflux mechanismrelating to cholesterol accumulation, including, modulation of asphingolipid enzyme to reduce cellular cholesterol accumulation. Themethod can also include interference with a cellular cholesterolsynthetic pathway.

Preferably, a useful compound of the subject method is a cyclodextrin ora derivative of a cyclodextrin. More preferably, the cyclodextrin orderivative thereof is hydroxypropyl beta cyclodextrin (HPβCD).

The subject invention further contemplates pharmaceutical compositionsuseful for treating, preventing or ameliorating a condition or symptomas described. Compositions according to the invention comprise, as anactive pharmaceutical ingredient (API), at least one cyclodextrin asdescribed herein and a vehicle pharmaceutically acceptable forinjectable administration. Preferably, a composition of the inventioncomprises hydroxyporpyl β cyclodextrin (HPβCD) or an analog, derivative,isomer or salt thereof.

Compositions of the invention can comprise two or more activeingredients, wherein the active ingredients can be two or more retinoidcompounds, or can include one or more retinoid compound in combinationwith a non-retinoid compound. The active ingredients can be administeredsequentially or concomitantly.

A preferable application of the subject method comprises treating,inhibiting or preventing a condition such as obesity, metabolicsyndrome, pre-diabetes, diabetes, diabetic kidney disease or conditionsor symptoms relating thereto in a patient suffering from or predisposedto such condition.

The method comprises administering the effective amount of the compound,such as a cyclodextrin or derivative of a cyclodextrin, by a commonroute of administration, such as intramuscular, intraperitoneal,intravenous (systemic), subcutaneous, transdermal, oral, rectal,inhalation, topical, and intranasal. A preferred route of administrationis subcutaneous.

Dosage ranges for administering a compound in accordance with a methodof the subject invention comprise from about 2-20 mg/kg/day to about4000 mg/kg three to five times per week (totaling up to about 20,000mg.kg/week), and can be administered at least two times per week up toabout three times per day.

Preferably, the method employs administering the compound as a singleactive ingredient, wherein more than one compound in the same class ofcompounds is considered a single ingredient. For example, administrationof HPβCD plus a derivative of HPβCD, both being in the class ofcyclodextrins, is considered a single active ingredient or singlecyclodextrin for purposes of the subject invention. Typically, thecompound is administered as a composition comprising the activeingredient and at least one pharmaceutically acceptable excipient orvehicle.

Alternatively, the method can employ the use of more than one activepharmaceutical ingredient, wherein the composition comprises two activeingredients, each from a different class of compounds where at least oneof the compounds is a cyclodextrin. For example, the method can compriseadministering a first active ingredient comprising one of morecyclodextrin or derivative thereof, and a second active ingredient whichis not a cyclodextrin or derivative thereof, and preferably apharmaceutically acceptable excipient or vehicle. For purposes of thesubject invention, a pharmaceutically acceptable excipient can include apreservative.

The second active ingredient can be selected from an antidiabetic agent,a cholesterol biosynthesis inhibitor, a cholesterol absorptioninhibitor, a bile acid sequestrant, niacin or niacin derivative, afibrate, a cholesteryl ester transferase protein, and an acetyl-coenzymeA acetyltransferase inhibitor or a biologic. Alternatively, the secondactive ingredient can be selected from an immunosuppressive agent,insulin, sulphonylurea, gliptin, metformin, thiazolidinedione, insulinsensitizer, incretin analogue, DPP4 inhibitor, VEG-interfering agent,growth factor, antinflammatory, vitamin D derivative, RAS systemblocker, and aldosterone blocker.

The method of the invention further includes treating, inhibiting,preventing, or ameliorating a condition, or a symptom or secondarycondition caused by accumulation of cholesterol in a cell or plasmamembrane of a cell. These conditions are listed above and incorporatedherein by reference. This embodiment of the invention can compriseadministering to a patient suffering from the condition, or symptom orsecondary condition, an effective amount of a compound which reducescellular accumulation of cholesterol by affecting a cellular influxmechanism or a cellular efflux mechanism relating to cholesterolaccumulation. The method can include modulation of a sphingolipid enzymeto reduce cellular cholesterol accumulation, and can further includeeffecting interference with a cellular cholesterol synthetic pathway.

In addition, the subject invention comprises a composition for treating,inhibiting, preventing, or ameliorating a symptom or related conditioncaused by, cellular accumulation of cholesterol, wherein the compositioncomprises an effective amount of at least one cyclodextrin or derivativeof cyclodextrin, and a pharmaceutically acceptable excipient or vehicle.

The composition can further comprises a second active pharmaceuticalingredient which is not a cyclodextrin or a derivative of acyclodextrin. The second active ingredient can be an antidiabetic agent,a cholesterol biosynthesis inhibitor, a cholesterol absorptioninhibitor, a bile acid sequestrant, niacin or niacin derivative, afibrate, a cholesteryl ester transferase protein, and an acetyl-coenzymeA acetyltransferase inhibitor or a biologic.

Alternatively, the second active ingredient can be an immunosuppressiveagent, insulin, sulphonylurea, gliptin, metformin, thiazolidinedione,insulin sensitizer, incretin analogue, DPP4 inhibitor, VEG-interferingagent, growth factor, antinflammatory, vitamin D derivative, RAS systemblocker, and aldosterone blocker.

In commercial use, the compound, composition and method of using thecomposition is useful in connection with a product or article ofmanufacture comprising the composition, as contained in a container,wherein the container is packaged with written instruction for using thecompound or composition in accordance with a method for treating acondition caused by, or symptom resulting from, cellular accumulation oflipid, as described herein.

A preferred article of manufacture comprises a compound or compositionof the invention, and written instruction indicating its use in treatingcellular cholesterol accumulation, or a condition or symptom associatedwith cellular cholesterol accumulation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows results of experiments illustrating CD improves diabetes invivo, specifically, 1-Cholesterol was depleted by subcutaneous injectionof hydroxypropyl-beta-cyclodextrin (CD) reverting diabetes and obesityin experimental animals and improving human islet cell function ex vivo.

FIG. 2 shows results of experiments illustrating CD protects fromDiabetic Kidney Disease in vivo, specifically, 2-Cholesterol wasdepleted by subcutaneous injection of Hydroxypropyl-beta-cyclodextrin(CD) and improves albuminuria, kidney weight and histological featuresof diabetic kidney disease.

FIG. 3 is a Table summarizing the safety of CD in BTBR mice treated forfive months (from the age of 4 weeks to the age of 24 weeks) withsubcutaneous administration of three weekly injections ofhydroxypropyl-β-cyclodextrin, specifically illustrating that3-Cholesterol was depleted by subcutaneous injection ofHydroxypropyl-beta-cyclodextrin (CD) and was safe even when administeredat very high doses of 4,000 mg/kg body weight.

FIG. 4 illustrates that 4-cholesterol accumulation occurs in podocytesexposed to the sera of patients with Diabetes and Diabetic KidneyDisease (DKD+), when compared to normal healthy controls and to patientswith Diabetes without Kidney Disease (DKD−).

FIG. 5 shows that CD protects podocytes from changes observed afterexposure to DKD+ sera, specifically illustrating that cholesteroldepletion with CD prevents several phenotypic changes, such as cellblebbing, cholesterol accumulation and cell apoptosis, observed innormal kidney cells (human podocytes) exposed to the sera of patientswith diabetic kidney disease.

FIG. 6 shows expression of sphingolipid related enzymes, e.g., SMPDL3b,are increased in diabetes in target organs such as the kidney and causeslipid dependent damage.

DETAILED DESCRIPTION OF THE INVENTION

It is known that elevated cholesterol levels, and in particularlow-density lipoprotein (LDL) cholesterol, in the plasma play animportant role in the development of microvascular and macrovascularcomplications of diabetes. Currently available treatments to lowercholesterol levels in patients, however, aim on lowering plasma LDL byblocking the synthesis of cholesterol in the liver (statins), bypreventing reabsorption of cholesterol into the circulatory system (bileacid resins, cholesterol absorption inhibitors), or by increasing HDLcholesterol (fibrates, niacin derivatives, cholesteryl ester transferprotein [CETP] inhibitor). None of the currently used medications aimson lowering plasma membrane or cellular cholesterol, except for liver xreceptor agonists (LXR), which have not yet been found to be safe inhumans.

One embodiment of the invention relates to metabolic disorders which canbe selected from a list comprising: diabetes (type 1 or type 2),prediabetes, obesity, metabolic syndrome, diabetic nephropathy, diabetickidney disease, diabetic neuropathy, diabetic retinopathy, diabetesrelated microvascular complication, diabetes related macrovascularcomplications, atherosclerosis, peripheral vascular disease, coronaryartery diseases, congestive heart failure, cardiac hypertrophy,myocardial infarction, endothelial dysfunction and hypertension, stroke,cerebrovascular accident, myocardial infarction, heart attack,cardiovascular accidents, fatty liver, steatohepatitis, NASH, insulinresistance.

In another embodiment of the invention, the drug used to prevent, treat,cure or reverse renal-related disorders is any drug that lowers theplasma membrane and cellular cholesterol content of the cell.

The drug can hereby be administered to an individual in a variety ofways. Routes of administration include, intramuscular, intraperitoneal,intravenous (systemic), subcutaneous (alone or in combination with drugsthat facilitate administration of relatively large volume ofsubcutaneous drugs), transdermal, oral, rectal, inhaled, topical, andintranasal routes. The drug can be administered together with otherbiologically active agents or components as pharmaceutically acceptablecarriers, diluents, excipients and vehicles.

A cyclodextrin described herein can be formulated into a pharmaceuticalcomposition. In one aspect, a compound can be combined with at least onepharmaceutically-acceptable carrier to produce a pharmaceuticalcomposition, prepared using techniques known in the art. In one aspect,a composition is prepared by admixing the compound with apharmaceutically-acceptable carrier. Depending upon the components to beadmixed, the components may or may not chemically or physically interactwith one another.

Compositions provided herein may be formulated to include at least onecyclodextrin, together with one or more pharmaceutically acceptablecarriers, including excipients, such as diluents, binders and the like,and can further include additives, such as stabilizing agents,preservatives, solubilizing agents, and buffers, as desired.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like, in additionto the active ingredient. Pharmaceutical compositions may also includemore than one active ingredient, such as a retinoid compound accordingto the invention, and one or more antimicrobial agents, antiinflammatoryagents, anesthetics, or the like. Pharmaceutically-acceptable carriersare known to those skilled in the art. These most typically would bestandard carriers for administration to humans, including solutions suchas sterile water, saline, and buffered solutions at physiological pH.

The pharmaceutical composition may be formulated for administration in anumber of ways depending on whether local or systemic treatment isdesired, or depending on the area to be treated. Administration can betopical, including ophthalmically, vaginally, rectally, intranasally,applied to the surface of the skin, or the like, as would be readilyunderstood by persons of ordinary skill in the art.

In practical use, a provided compound of the present invention can becombined as the active ingredient in an admixture with a pharmaceuticalcarrier or vehicle according to conventional pharmaceutical compoundingtechniques. The carrier used can forms depend on the dosage form desiredfor administration, for example, oral, parenteral (includingintravenous), urethral, vaginal, nasal, dermal, topical, transdermal,pulmonary, deep lung, inhalation, buccal, sublingual administration, orthe like.

If in an aqueous solution, as preferred, a provided cyclodextrincomposition may be appropriately buffered by means of saline, acetate,phosphate, citrate, acetate or other buffering agents, which may be atany physiologically acceptable pH, generally from about pH 4 to about pH7. A combination of buffering agents may also be employed, such asphosphate buffered saline, a saline and acetate buffer, and the like. Inthe case of saline, a 0.9% saline solution may be employed. In the caseof acetate, phosphate, citrate, acetate and the like, a 50 mM solutionmay be employed. In addition to buffering agents, a suitablepreservative may be employed, to prevent or limit bacteria and othermicrobial growth. One such preservative that may be employed is 0.05%benzalkonium chloride.

Preparations for administration include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of aqueous ornon-aqueous carriers include water, alcoholic/aqueous solutions,emulsions or suspensions, including saline and buffered media.Parenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's, or fixed oils.

Intravenous vehicles can be fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. It will beappreciated that the actual preferred amounts of active compound in aspecified case will vary according to the specific compound beingutilized, the particular compositions formulated, the mode ofapplication, and the particular situs and mammal being treated.

In preparing the compositions for oral dosage form, typicalpharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols, flavoring agents, preservatives, coloringagents and the like in the case of oral liquid preparations, such as,for example, suspensions, elixirs and solutions; or carriers such asstarches, sugars, microcrystalline cellulose, diluents, granulatingagents, lubricants, binders, disintegrating agents and the like in thecase of oral solid preparations such as, for example, powders, hard andsoft capsules and tablets.

For solid administration formulations, any of a variety of thickening,filler, bulking and carrier additives may be employed, such as starches,sugars, fatty acids and the like. Formulation excipients may includepolyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethyleneglycol, mannitol, sodium chloride and sodium citrate. For injection orother liquid administration formulations, water containing at least oneor more buffering constituents is preferred, and stabilizing agents,preservatives and solubilizing agents may also be employed.

It is also contemplated that provided compounds of the present inventionmay be in a dried and particulate form. In certain embodiments, theparticles are between about 0.5 and 6.0 μm, such that the particles havesufficient mass to settle on the lung surface, and not be exhaled, butare small enough that they are not deposited on surfaces of the airpassages prior to reaching the lung. Any of a variety of differenttechniques may be used to make dry powder microparticles, including butnot limited to micro-milling, spray drying and a quick freeze aerosolfollowed by lyophilization.

For pharmaceutical formulations, it is also contemplated that any of avariety of measured-release, slow-release or time-release formulationsand additives may be employed, so that the dosage may be formulated soas to effect delivery of a provided compound over a period of time,commonly referred to as controlled, delayed, extended, slow, orsustained, release formulations. For example, gelatin, sodiumcarboxymethylcellulose and/or other cellulosic excipients may beincluded to provide time-release or slower-release formulations,especially for administration by subcutaneous and intramuscularinjection.

Provided cyclodextrins of the present invention may be therapeuticallyadministered by means of an injection, typically a subcutaneous orintramuscular injection, such as in the gluteal or deltoid muscle, of atime release injectable formulation. In one embodiment, a providedcompound of the present invention is formulated with a PEG, such aspoly(ethylene glycol) 3350, and optionally one or more additionalexcipients and preservatives, including but not limited to excipientssuch as salts, polysorbate 80, sodium hydroxide or hydrochloric acid toadjust pH, and the like.

The formulation may be such that an application, administration, orinjection is required on a daily, weekly, monthly or other periodicbasis, depending on the concentration and amount of a provided compound,the biodegradation rate of a polymer used in the formulation, and otherfactors known to those of skill in the art.

In another embodiment, the drug used to prevent, treat, cure or reverserenal-related disorders is Cyclodextrin or any of its derivatives oranalogs.

In another embodiment, Cyclodextrin is used to prevent, treat or reducehyperglycemia (fasting or postprandial) in patients.

The present disclosure also relates to methods reducing the plasmamembrane or cellular cholesterol content in any cells of any organ as atool to prevent, treat, cure or reverse obesity, metabolic syndrome,pre-diabetes and diabetes related disorders.

In one embodiment, the plasma membrane or cellular cholesterol contentis reduced in any cell of the body as a tool to prevent, treat, cure orreverse obesity, metabolic syndrome, pre-diabetes and diabetes relatedkidney disorders. A compound or composition of the invention can employa cyclodextrin for treating, inhibiting, preventing, reducing, orreversing cholesterol accumulaton in a cell or plasma membrane of a cellof a central or peripheral organ affected by diabetes or which isresponsible for the development of diabetes.

In another embodiment, the plasma membrane or cellular cholesterolcontent is reduced in pancreatic beta cells to prevent, treat, cure orreverse impaired glucose stimulated insulin release, local or systemicinflammation and or the autoimmune attack to pancreatic islets.

In another embodiment, Cyclodextrin or its derivatives are used to atleast partially deplete cells from plasma membrane cholesterol alone orin combination with other drugs currently used or being studied for theprevention and the treatment of diabetes such as immunosuppressiveagents, insulin, sulphonylureas, gliptins, metofrmin, TZDs or anyinsulin sensitizers, incretin analogues and DPP4 inhibitors, agentsinterfering with VEGF and other growth factors, antinflammatorymedications, vitamin D derivatives, blockers of the RAS system and ofaldosterone.

In another embodiment, Hydroxypropyl Beta Cyclodextrin (HPβCD) is safewhen administered to mice at a dose ranging from about 2 to about 20mg/kg/day and can be administered in amounts up to about 4,000 mg/kgfrom three times to five times per week (totaling about 20,000mg/kg/week).

In another embodiment, Hydroxypropyl Beta Cyclodextrin (HP CD) is safewhen administered subcutaneously at doses as high as 4000 mg/kg threetimes a week.

In another embodiment, Cyclodextrin, its derivatives, or any otherplasma membrane or cellular cholesterol lowering drug is used incombination with a cholesterol biosynthesis inhibitor, such as a HMG-CoAreductase inhibitor. HMG-CoA reductase inhibitor drugs include drugssuch as Simvastatin, Atorvastatin, Lovastatin, Rosuvastatin, Mevastatin,Pravastatin, Fluvastatin, Pitavastatin, Rosuvastatin, Cerivastatin,Rivastatin, Itavastatin, or ZD-4522.

In another embodiment, Cyclodextrin, its derivatives, or any otherplasma membrane or cellular cholesterol lowering drug is used incombination with a cholesterol absorption inhibitor, such as aEzetimibe, SCH-48461.

In another embodiment, Cyclodextrin, its derivatives, or any otherplasma membrane or cellular cholesterol lowering drug is used incombination with bile acid sequestrants and resins (Colestipol,Colestilan, Colextran, Cholestyramine, Colesevelam).

In another embodiment, Cyclodextrin, its derivatives, or any otherplasma membrane or cellular cholesterol lowering drug is used incombination with Niacin and Niacin derivatives such as Niceritrol,Nicofuranose, Niacin, Nicotinyl alcohol, Acipimox.

In another embodiment, Cyclodextrin, its derivatives, or any otherplasma membrane or cellular cholesterol lowering drug is used incombination with fibrates such as Fenobrate, Simfibrate, Ronifibrate,Ciprofibrate, Clinofibrate, Clofibride, Etofibrate, Clofibrate,Bezafibrate, Aluminium clofibrate, Gemfibrozil.

In another embodiment, Cyclodextrin, its derivatives, or any otherplasma membrane or cellular cholesterol lowering drug is used incombination with cholesteryl ester transfer protein (CETP) inhibitorssuch as Dalcetrapib, Torcetrapib, Anacetrapib.

In another embodiment, Cyclodextrin, its derivatives, or any otherplasma membrane or cellular cholesterol lowering drug is used incombination with Acetyl-Coenzyme A acetyltransferase (ACAT) inhibitors(such as avasimibe) or microsomal triglyceride transport inhibitors.

These combination treatments may also be effective for the treatment orcontrol of one or more obesity, metabolic syndrome, pre-diabetes anddiabetes related disorders selected from the group consisting ofatherosclerosis, insulin resistance, hyperlipidemia,hypertriglyceridemia, dyslipidemia, high LDL, and low HDL.

EXAMPLES

Experimental results showed increased cholesterol in association withdown-regulation of ATP-binding cassette transporter ABCA1 occurs innormal human podocytes exposed to the sera of patients with type1-diabetes and albuminuria (DKD+) when compared to diabetic patientswith normoalbuminuria (DKD−) and similar duration of diabetes and lipidprofile. Glomerular down-regulation of ABCA1 was confirmed in biopsiesfrom patients with early DKD (n=70) when compared to normal livingdonors (n=32).

Induction of cholesterol efflux with cyclodextrin (CD) but notinhibition of cholesterol synthesis with simvastatin prevented podocyteinjury observed in vitro after exposure to patient sera. Subcutaneousadministration of CD to diabetic BTBR-ob/ob mice was safe and reducedalbuminuria, mesangial expansion, kidney weight and cortical cholesterolcontent. This was followed by an improvement of fasting insulin, bloodglucose, body weight, glucose tolerance in vivo and improved glucosestimulated insulin release in human islets in vitro.

Our data suggest that impaired reverse cholesterol transportcharacterizes clinical and experimental DKD and negatively influencespodocyte function. Treatment with CD is safe and effective in preservingpodocyte function in vitro and in vivo, in ameliorating human pancreaticbeta cells ex vivo and in improving the metabolic control of diabetes invivo

We used a previously established cell-based assay in whichdifferentiated human podocytes are exposed to 4% patient sera for 24hours to identify new pathways and targets in DKD. Podocytes exposed tothe sera of patients with DKD showed increased cholesterol accumulationin association with down-regulation of ABCA1 expression that wasindependent of circulating cholesterol. Excessive cholesterol depositionhas indeed been described in glomeruli of rodent models of T1D and T2Dand may contribute to DKD development and progression.

Research Design And Methods

Patient sera and kidney biopsies. Serum samples were obtained from tenhealthy controls and twenty patients with T1D from the Finnish DiabeticNephropathy Study (FinnDiane). T1D was defined as onset of diabetesbefore 40 years of age and permanent insulin treatment initiated within1 year of diagnosis. Urinary albumin excretion rate (AER) was defined asnormal AER (<30 mg/24 h), microalbuminuria (30<300 mg/24 h), andmacroalbuminuria (≧300 mg/24 h). Fasting glucose values were measuredusing a Hemocue device (Hemocue, Finland). Serum lipids were determinedwith a Konelab analyser (Thermo Scientific, Finland). Other biochemicalanalyses were performed in an accredited hospital laboratory (HUSLAB,Helsinki). For glomerular mRNA expression profiles, kidney biopsyspecimens were procured from 70 Southwestern American Indians afterobtaining informed consent. Human renal biopsies from pre-transplant,healthy living donors (n=32), membranous nephropathy (n=21) and focalsegmental glomerulosclerosis (n=18) patients were obtained from theEuropean Renal cDNA Bank.

Microarray analysis and PCR. For in vitro experiments, Illuminatechnology was utilized to study mRNA expression in human podocytesexposed to four independent pools of sera from 2-3 patients each(control, DKD− and DKD+). Glomerular mRNA expression profiles wereperfomed with Affymetrix GeneChip arrays as described.

Human podocyte culture. Human podocytes were cultured and differentiatedas previously described and serum starved in 0.2% FBS prior toexperiments. When patient sera were utilized, starved cells were exposedto 4% patient sera for 24 hours. For insulin treatment experiments, 100nmol insulin was added to the culture medium for 15 minutes afterexposure to patient sera. For cyclodextrin or statin treatmentexperiments serum starved human podocytes were pretreated for 1 hourwith 5 mM Methyl-beta-Cyclodextrin (CD, Sigma) or simvastatin (1 μM,Sigma).

Immunofluorescence staining. Cells cultured in chamber slides were fixedwith 4% PFA for 30 minutes at 37° C. and permeabilized with 0.1% TritonX-100, followed by exposure to mouse-anti-phospho-Caveolin (pY14, BDBiosciences), anti-active-RhoA (New East Biosciences), or anti-vimentin(Sigma) antibodies. Fluorescence detection was performed using AlexaFluor secondary antibodies (Invitrogen). For cholesterol determination,filipin (Sigma) staining was performed as described. F-actin wasvisualized by Rhodamine Phalloidin (Invitrogen). Two hundred consecutivecells per condition were studied. Slides were prepared with DAPIenriched mounting media (Vectashield) and analyzed by confocalmicroscopy.

Apoptosis analysis. Apoptosis was assessed using the Caspase-3/CPP32Colorimetric Assay kit (Biovision) according to the manufacturer'sdescription. Caspase 3 activity was normalized to cell number andexpressed as fold change to controls.

Determination of cholesterol content. Esterified cholesterol wasdetermined as difference between total and free cholesterol using anenzymatic assay and normalized to cell protein content. The cellularcontent of lipid droplets was determined using Oil red O (ORO). Cellswere fixed and permeabilized as described above, washed in PBS and in60% isopropanol, followed by incubation in ORO (0.5% ORO in isopropanol,1:3 diluted) for 15 min at room temperature and counterstaining withhematoxylin for 1 min. The fraction of ORO positive cells over twohundred consecutive cells was calculated by bright field microscopy.

Western blotting and luminex. Cell lysate collection and Westernblotting was performed as previously described. The following primaryantibodies were used: rabbit-polyclonal-anti-MyD88 (Cell Signaling),rabbit-polyclonal-anti-phosphorylated (Y473) or anti-total-AKT (CellSignaling), mouse-monoclonal-anti-rhoA (Santa Cruz), ormouse-monoclonal-anti-Gapdh (6C5, Calbiochem) antibody. SecondaryAnti-mouse-IgG-HRP or and Anti-rabbit-IgG-HRP Conjugate (Promega) wereused. Image acquisition was performed using the chemiluminescent imagerSRX-101A (Konica Minolta medical imaging, USA) and band densitometry wasanalyzed using ImageJ software (NIH). Alternatively,phosphorylated/total AKT was quantified using luminex technology aspreviously reported.

Quantitative Real Time PCR (QRT-PCR). Podocyte RNA was extracted usingthe RNAeasy Mini Kit (Qiagen). Reverse transcription was performed usingthe high-capacity cDNA reverse transcriptase kit (Applied Biosystems)according to the manufacturer's protocols. QRT-PCR was performed in theStepOnePlus real-time PCR system (Applied Biosystems) using thePerfeCTa® SYBR® Green FastMix (Quanta Biosciences). Relative geneexpression was determined as 2̂-ΔCt, with ΔCt being the differencebetween the cycle threshold (Ct) value of the target gene and Gapdh. Forsemi-quantitative expression analysis, PCR was performed and analyzed bygel electrophoresis. Amplification product intensities were determinedusing ImageJ software (NIH), values were normalized and expressed asfold changes in gene expression over GAPDH. The following primers wereused: hABCA1-F, AACAGTTTGTGGCCCTTTTG; hABCA1-R, AGTTCCAGGCTGGGGTACTT;hLDL-R-F, TCACTCCATCTCAAGCATCG; hLDL-R-R, GGTGGTCCTCTCACACCAGT;hHMG-CoA-R-F, GGCATTTGACAGCACTAGCA; hHMG-CoA-R-R, GCTGGAATGACAGCTTCACA;hGAPDH-F, GTCAGTGGTGGACCTGACCT; hGAPDH-R, Hs_ABCA1-SGQuantiTect-Primer-Assay (Qiagen); mAbca1-F, GGACATGCACAAGGTCCTGA;mAbca1-R, CAGAAAATCCTGGAGCTTCAAA.

BTBR ob/ob mice treatment and analysis. BTBR ob/ob mice were purchasedfrom Jackson Laboratories. Mice were injected subcutaneously with 4,000mg/kg CD or saline as reported previously, three times per week for 5months. Urine was collected, and body weight and glycemia (OneTouch)were determined weekly. Six mice per group were analyzed. All animalprocedures were approved by the Institutional Animal Care and UseCommittee (IACUC). After isotonic saline perfusion, the right kidney wasremoved for cholesterol content determination and mRNA extraction. Oneleft kidney pole was embedded in OCT, a second pole fixed in 4% PFA andparaffin-embedded for histological analysis. Blood samples were analyzedfor CBC, lipid panel, AST, ALT, Alkaline Phosphatase, GGT, and BUN inthe Comparative Laboratory Core Facility, University of Miami. Serumcreatinine was determined by tandem mass spectrometry at the UAB-UCSDO'Brien Core Center, University of Alabama at Birmingham, using themethods previously described. The urine albumin content was measured byELISA (Bethyl Laboratories). Urinary creatinine was assessed by an assaybased on the Jaffe method (Stanbio). Values are expressed as pgalbumin/mg creatinine. Fasting plasma insulin was determined by ELISA(Mercodia, SW). Intraperitoneal glucose tolerance tests (IPGTT) wereperformed 4 months after treatment; after 5-hr fasting, blood glucosewas recorded at baseline and up to 180 minutes after a glucose bolus(1.5 g/kg). For insulin sensitivity, glycemia was monitored at baselineand up to 150 minutes after intraperitoneal injection of 4 mU/g of shortacting insulin. Human islets from four different isolations werepretreated with 0.5 mM CD for one hour and perfused as described beforeto determine insulin release in response to glucose and KCl.

Histology, assessment of mesangial expansion and glomerular surfacearea. Periodic acid-Shiff's (PAS) staining of 4 μm thick tissue sectionswas performed. Twenty glomeruli per section were analyzed for mesangialexpansion by semiquantitative analysis (scale 0-4) performed by twoblinded independent investigators. The glomerular surface was delineatedin each encountered glomerulus and the mean surface area calculated asdescribed.

SMPDL3b western blot was performed as we have previously reported.SMPDL3b overexpressing and knock down cell lines were also previouslyreported and are now utilized for the determination of cholesterolcontent and RhoA expression as described above.

Statistical analysis. Data are shown as mean and standard deviations.Four to 8 independent experiments were performed for in vitro studies.Six mice per group were used for in vivo experiments. Statisticalanalysis was performed with one-way ANOVA. When one-way ANOVA showedstatistical significance, results were compared using t-test afterTukey's correction for multiple comparisons. Results were consideredstatistically significant at p<0.05.

RESULTS

Clinical laboratory measurements and patient population. We studied 30male subjects divided into three groups based on clinicalcharacteristics at the time of collection of the sera samples. The studysubjects included:

1) 10 patients with T1D, normoalbuminuria and normal creatinine, definedas patients without diabetic kidney disease (DKD−),

2) 10 patients with T1D, albuminuria and altered creatinine, defined aspatients with diabetic kidney disease (DKD+),

3) 10 healthy controls (C).

The three groups were not significantly different for age, totalcholesterol, HDL-, LDL-cholesterol and triglycerides. All patients weretaking agents to block the renin-angiotensin-aldosterone system.Duration of diabetes, fasting plasma glucose and HbA1C were notsignificantly different among DKD+ and DKD− patients. The mean estimatedglomerular filtration rate (eGFR) was 101 ml/min/1.73 m2 in DKD−, 97ml/min/1.73 m2 in C and 43 ml/min/1.73 m2 in the DKD+ group. Seracollected seven years prior from five of the patients with DKD (meaneGFR 98 ml/min/1.73 m2) was utilized in selected experiments.

Podocytes exposed to DKD+ sera have a characteristic cDNA signature. RNAwas extracted from differentiated podocytes cultured in the presence ofpatient sera as we previously reported. cDNA microarray analysisrevealed that main pathways altered in DKD+ treated when compared toDKD− treated podocytes included genes involved in actin remodeling,insulin signaling, cytokine signaling (primarily through TLR4, TNF□ andIL1□), and apoptosis. We validated these findings at the protein leveldemonstrating by western blotting that in DKD+ treated podocytes, MyD88expression was increased, the ability of insulin to phosphorylate AKTwas impaired and the amount of cleaved caspase 3 was increased.

Normal human podocytes exposed to sera of patients with DKD exhibit cellblebbing. Podocytes exposed to the sera of patients with DKD experiencedpronounced actin cytoskeleton remodeling with localized decoupling ofthe cytoskeleton from the plasma membrane (blebbing), which was evidentin both the phalloidin staining (F-actin) and the brightfield images andwhich was very different from what we have reported in focal andsegmental glomerulosclerosis. Quantitative analysis of cell blebbing(percentage of cells with blebs out of a total of 200 consecutive cellsanalyzed) revealed this phenotype in 80% of cells exposed to DKD+ sera,but in only 20% of cells exposed to DKD− sera and 5% in the controls.Cell blebbing was accompanied by the redistribution of active RhoA atthe site of cell blebbing and by an increase in total RhoA. Cellblebbing was not a consequence of reduced GFR in the DKD+ group, ashistorical sera collected from five of the patients with T1D and normalGFR that ultimately developed DKD caused the same degree of cellblebbing in cultured podocytes as the sera from the same patientscollected on average 7□2 years later, when the patients had establishedDKD with macroalbuminuria and low GFR.

Impaired reverse cholesterol transport in podocytes exposed to DKD+ seraand in glomeruli from patients with early diabetes. As inflammation,insulin resistance, apoptosis and cytoskeleton remodeling are linked bythe intracellular accumulation of lipids in the pathogenesis ofnon-alcoholic steatohepatitis (NASH syndrome), and accumulation ofcholesterol has been described in the cortex of the kidneys of mice withDKD, we explored if podocytes cultured in the presence of sera ofpatients with DKD accumulate intracellular cholesterol. We were able todemonstrate an increased number of ORO and filipin positive cells inDKD− and DKD+ treated cells, more so in the DKD+ treated cells.Quantitative analysis of total, free and esterified cholesterol revealedsignificantly increased esterified cholesterol in DKD+ treated cellswhen compared to cells treated with sera from control subjects. Thisincrease was likely due to impaired cholesterol efflux, as LDL-receptorand HMG-CoA reductase expression were unchanged, while ABCA1 mRNAexpression was down-regulated in DKD+ treated podocytes. We then studiedthe mRNA expression of lipid related genes in glomeruli from additional70 patients with T2D and early DKD when compared to 32 normal livingdonors, and demonstrated significant down-regulation of ABCA1 in DKD.Interestingly, down-regulation of ABCA1 mRNA expression was a feature ofDKD only, as ABCA1 was not regulated in other proteinuric glomerulardiseases such as membranous nephropathy (MN, n=21) and focal segmentalglomerulosclerosis (FSGS, n=18).

Cyclodextrin protects podocytes in vitro. As the exposure of podocytesin culture to DKD+ sera caused accumulation of total cholesterol inassociation with decreased ABCA1 expression, a transporter responsiblefor cholesterol efflux, we went on to test the hypothesis thatcyclodextrin would protect podocytes from the actin cytoskeletonremodeling and cell blebbing observed after exposure to the sera frompatients with DKD. We were able to demonstrate that CD reduced thenumber of filipin-positive cells and preserved the localization ofphosphorylated caveolin to focal adhesion sites. Quantitativecholesterol analysis showed that CD prevented the accumulation of totaland esterified cholesterol in DKD+ treated podocytes. Furthermore,prevention of intracellular cholesterol accumulation with CD alsoprevented DKD+ induced apoptosis, insulin resistance and MyD88expression. Blockade of HMG-CoA reductase with simvastatin in podocytesdid not protect from DKD+ induced actin cytoskeleton remodeling.

Subcutaneous administration of CD protects BTBR ob/ob mice from thedevelopment of DKD. BTBR (black and tan, brachiuric) ob/ob (leptindeficient) mice have been described as a mouse model of progressive DKD.After establishing a dose and a mode of administration based onpreliminary toxicology studies, we treated 4-week old BTBR mice withsubcutaneous administration of three weekly injections ofhydroxypropyl-β-cyclodextrin (CD, 4,000 mg/kg body weight) for fivemonths. Although no changes in albumin excretion rates were observed upto 2 months after treatment initiation in homozygous mice, at 3 months asignificant down-regulation of the albumin/creatinine ratios wasobserved in the morning spot urine samples (p<0.001) in CD treated whencompared to untreated BTBR ob/ob mice. This decrease persisted untilsacrifice (5 months after initiation of treatment. At sacrifice, CDtreated mice showed a reduction of kidney weight. CD did not affectABCA1 mRNA expression in the kidney cortex but resulted in a significantreduction of total cholesterol. Blood urea nitrogen (BUN) and creatininewere not significantly affected by CD treatment. However, CD treatmentresulted in a reduction of mesangial expansion without affecting theglomerular surface area.

After four months of treatment, we also observed a reduction of bodyweight, which was accompanied by a concomitant improvement of randomglycemia. Furthermore, sera collected at sacrifice demonstrated asignificant improvement of fasting plasma insulin and fasting plasmaglucose. IPGTT performed one week prior to sacrifice were improved in CDtreated homozygous mice when compared to homozygous controls. Asimprovement was observed despite a similar insulin sensitivity test, weanalyzed the effect of low dose CD on the function of four differentpreparations of human islet cells. A significant improvement in glucosestimulated insulin release was observed in CD treated human islets whencompared to untreated human islets. To determine whether the beneficialeffect of CD on islet cell function was associated with the modulationof ABCA1 expression in pancreatic islets, we performedimmunofluorescence staining using a rabbit polyclonal ABCA1 antibody(gift from Dr. A. Mendez) and determined ABCA1 expression as meanfluorescence intensity per pancreata analyzed. Pancreata from homozygousBTBR ob/ob mice were characterized by significantly decreased ABCA1expression when compared to heterozygous littermates (p<0.001) and CDtreatment significantly increased ABCA1 expression in pancreata ofhomozygous BTBR ob/ob (p<0.001) and heterozygous BTBR ob/+ mice(p<0.01). As hemolytic anemia and liver toxicity have been describedwhile using other cyclodextrin derivatives in rodents and humans, andbecause we administered high dose CD for a period of 5 months, westudied hemoglobin, aspartate aminotransferase (AST), alaninetransaminase (ALT) and gamma-glutamyltransferase (GGT) at sacrifice. Noabnormalities due to chronic CD administration were observed indicatingthat the chronic use of CD is not accompanied by adverse side effects(see Table).

The efficacy and/or safety of the subject invention can be illustratedby reference to the figures appended hereto, the legends for which areprovided below:

FIG. 1 shows a series of panels, (a) through (i) illustrating results ofexperiments or studies conducted which support that CD improves diabetesin vivo. Specifically referring to each of the panels of FIG. 1, (a,b)CD administered to homozygous and heterozygous BTBR ob/ob micesubcutaneously three times a week (n=6 per group) resulted in asignificant reduction in body weight (mean±SD) starting at four monthafter the initiation of the treatment. *p<0.05, “p<0.01. (c) CDadministered to homozygous BTBR ob/ob mice resulted in a significantreduction in random glycemia (mean±SD) starting at four month after theinitiation of the treatment. (d,e) Bar graph analysis (mean±SD) offasting plasma insulin and glucose concentrations. Fasting plasmainsulin (**p<0.01; ***p<0.001)(d) and fasting plasma glucose(”p<0.01)(e) were significantly increased in homozygous mice whencompared to heterozygous controls. The increase was prevented by CDtreatment (p<0.01). (f) IPGTT performed at 5 month after the initiationof the CD treatment showed improved glucose tolerance in CD treated BTBRob/ob mice when compared to untreated BTBR ob/ob mice. (g) CD treatmentdid not affect the sensitivity to a single dose of short acting insulin(4 mU/g) in BTBR ob/ob mice. (h) Representative perifusion experimentand bar graph analysis of the area under the curve (AUC) demonstratingthe effect of 0.5 mM CD on glucose stimulated insulin release in humanpancreatic islets from four independent donors (**p<0.01). (i)Immunofluorescence staining for ABCA1 reveals increased ABCA1 expressionin pancreata of CD treated BTBR ob/ob mice when compared to untreatedlittermates (FIG. 6 i, left). Bar graph analysis (FIG. 6 i, right)showing that pancreata isolated from homozygous BTBR ob/ob mice arecharacterized by significantly decreased ABCA1 expression when comparedto heterozygous littermates (###p<0.001). CD treatment significantlyincreased ABCA1 expression in pancreata of homozygous BTBR ob/ob(***p<0.001) and heterozygous BTBR ob/+ mice (**p<0.01).

FIG. 2 shows panels (a) through (i) supporting that CD protects fromDiabetic Kidney Disease in vivo. (a) CD administered to homozygous andheterozygous BTBR ob/ob mice subcutaneously three times a week (n=6 pergroup) resulted in a reduction in albumin/creatinine ratios (mean±SD)starting at 3 months after the initiation of the treatment. *p<0.05,**p<0.01, ***p<0.001. (b) Kidney weight (mean±SD) was significantlyincreased in homozygous mice (***p<0.001), and such an increase wasprevented by CD treatment (##p<0.01). (c) Bar graph analysis (mean±SD)of the effect of CD on ABCA1 mRNA expression in kidney cortexes ofhomozygous and heterozygous BTBR ob/ob mice. (d) Bar graph analysis(mean±SD) of the effect of CD on the total cholesterol content in kidneycortexes of homozygous and heterozygous BTBR ob/ob mice. (e, f) Bargraph analysis (mean±SD) showing that serum BUN and creatinineconcentrations remain unchanged after CD treatment of the mice.Measurements were performed on serum obtained from the mice atsacrifice. (g) Representative PAS staining of kidney sections fromhomozygous and heterozygous BTBR ob/ob mice after five months oftreatment with either CD or vehicle. (h, i) Bar graph analysis (mean±SD)of the scores for mesangial expansion (h) and of the glomerular surfacearea (i) on PAS stained kidney sections from homozygous and heterozygousBTBR ob/ob mice after five months of treatment with either CD or vehiclewere assessed by two blinded, independent investigators. *p<0.05 whencomparing DKD+ versus C. #p<0.05 when comparing CD treated versusuntreated mice.

FIG. 3 is a table illustrating the safety of CD in 4-week old BTBR micetreated following subcutaneous administration of three weekly injectionsof hydroxypropyl-β-cyclodextrin (CD, 4,000 mg/kg body weight) for fivemonths. As older and more toxic CD derivatives have been shown to causeanemia and liver toxicity, we tested the effect of long-termadministration of high doses hydroxypropyl-β-cyclodextrin in diabeticand control mice.

In FIG. 4( a) through (i), results are shown that cholesterolaccumulation occurs in podocytes exposed to DKD+ sera (a) RepresentativeOil Red O staining of podocytes exposed to DKD+ sera when compared to Cand DKD− sera. Black arrows point to spots of major lipid dropletaccumulation. (b) Representative filipin staining (orange) andphosphorylated caveolin staining (green) of podocytes exposed to DKD+sera when compared to C and DKD−. (c) Bar graph quantitative analysis(mean±SD) of Oil Red O positive cells in podocytes exposed to the poolsof sera from 10 patients with DKD−, DKD+ or to pools of the sera fromcontrols, demonstrating that exposure to both, DKD− and DKD+ sera, causesignificant lipid droplet accumulation in cultured human podocytes.*p<0.05, ***p<0.001. (d, e, f) Bar graph analysis (mean±SD) of totalcholesterol (Tot C), free cholesterol (Free C) and esterifiedcholesterol (Est C) as determined via enzymatic reaction in podocytesexposed to pools of DKD+ sera when compared to C and DKD−. *p<0.05. (g,h, i) Quantitative RT PCR analysis (mean±SD) of LDL receptor, HMG-CoAreductase and ABCA1 expression in podocytes exposed to individualpatient sera. ***p<0.001. (j). Transcriptional analysis of glomerulargene expression of lipid related genes in 70 patients with early DKD, 21patients with membranous nephropathy (MN) and 18 patients with focalsegmental glomerulosclerosis (FSGS) when compared to 32 living donors.Numbers reflect fold change in disease when compared to living donors.

FIG. 5 panels (a) through (f) show that. CD protects podocytes fromchanges observed after exposure to DKD+ sera. (a) Representativephalloidin (red) and phosphorylated caveolin (green) confocal images ofnormal human podocytes exposed to DKD+ sera when compared to C and DKD−sera in the presence (CD) or absence (control) of CD. DAPI (blue) wasused to identify nuclei (b, c) Bar graph analysis (mean±SD) of theeffect of CD on total (B) and esterified cholesterol (C) in CD treated(+) versus untreated (−) podocytes exposed to DKD+ sera when compared toC and DKD− sera. *p<0.05 when comparing DKD+ versus C. #p<0.05 whencomparing CD treated versus untreated podocytes in the same group. (d,e, f) Bar graph analysis (mean±SD) of cleaved Caspase 3, insulinstimulated AKT phosphorylation and MyD88 expression in CD treated (+)versus untreated (−) podocytes exposed to DKD+ sera when compared to Cand DKD− sera. *p<0.05 and **p<0.01 when comparing DKD+ versus C.#p<0.05 when comparing CD treated versus untreated podocytes in the samegroup.

FIG. 6 panels (a) through (f) illustrates that expression ofsphingolipid related enzymes, i.e. SMPDL3b, are increased in diabetes intarget organs such as the kidney and causes lipid dependent damage. (a)Transcriptional analysis of glomerular gene expression of SMPDL3b in 12patients with DKD when compared to 32 living donors. Numbers reflectfold change in disease when compared to living donors. *p<0.05 (b)Representative western blot and relative bar graph analysis for SMPDL3bprotein expression in normal human podocytes cultured in the presence ofsera from healthy controls (C) or age and sex matched diabetic patientswith DKD (DKD+) or diabetic patients without DKD (but with the samediabetes duration and plasma lipid profile). p<0.05 (c, d)Representative Oil Red O staining and bar graph analysis of Oil Red O,Cholesterol, Trygliceride and Phospholipids in normal human podocytesand in podocytes overexpressing SMPDL3b (SMPDL3b OE) cultured in normalmedia (control) or in media supplemented with TNFα. *p<0.05 whencomparing TNFα treated or SMPDL3b OE to controls. ### p,0.001 whencomparing Oil Red O staining in SMPDL3b OE versus empty vector (EV)control (□) Representative phalloidin staining demonstrating actincytoskeleton remodeling in normal human podocytes exposed to DKD+ sera,a phenomenon that was prevented in cells where SMPDL3b was silenced(SMPDL3b KD). (f) Representative western blot and bar graph analysisdemonstrating that SMPDL3b KD human podocytes are resistant to theincreased RhoA expression observed after exposure to DKD+ sera. *p<0.05.

CONCLUSIONS

2-Hydroxypropyl-β-cyclodextrin (2-HPβCD), a strong cholesterol acceptor,is an effective way to sequester cholesterol and to protect any cellaffected by diabetes, prediabetes, metabolic syndrome and obesity fromcholesterol dependent damage in vivo and in vitro.

It is recognized that related inventions may be within the spirit of thedisclosures herein. Also no omission in the current application isintended to limit the inventors to the current claims or disclosures.While certain preferred and alternative embodiments of the inventionhave been set forth for purposes of disclosing the invention,modifications to the disclosed embodiments may occur to those who areskilled in the art.

1. A method for reducing lipid content in a cell or plasma membrane of acell in a patient suffering from a condition selected from diabetes(type 1), diabetes (type 2), prediabetes, obesity, metabolic syndrome,diabetic nephropathy, diabetic kidney disease, diabetic neuropathy,diabetic retinopathy, diabetes related microvascular complication,diabetes related macrovascular complications, atherosclerosis,peripheral vascular disease, coronary artery diseases, congestive heartfailure, cardiac hypertrophy, myocardial infarction, endothelialdysfunction and hypertension, stroke, cerebrovascular accident,myocardial infarction, heart attack, cardiovascular accidents, fattyliver, steatohepatitis, NASH, and insulin resistance, said methodcomprising administering to the patient an effective amount of acompound which reduces cellular lipid content.
 2. The method of claim 1wherein the compound inhibits a cellular influx mechanism or increases acellular efflux mechanism relating to cholesterol accumulation.
 3. Themethod of claim 1 wherein the compound modulates a sphingolipid enzymeto reduce cellular cholesterol accumulation.
 4. The method of claim 1wherein the cell is in a central or peripheral organ affected by orresponsible for development of diabetes.
 5. The method of claim 4wherein the organ is a pancreas.
 6. The method of claim 2 wherein thecompound further interferes with a cellular cholesterol syntheticpathway.
 7. The method of claim 1 wherein the compound is a cyclodextrinor a derivative or analog of a cyclodextrin.
 8. The method of claim 7wherein the cyclodextrin is hydroxypropyl beta cyclodextrin.
 9. Themethod of claim 1 wherein the condition is selected from obesity,metabolic syndrome, pre-diabetes, diabetes, diabetic kidney disease orconditions or symptoms relating thereto.
 10. The method of claim 7wherein the cyclodextrin or derivative of a cyclodextrin is administeredby a route selected from intramuscular, intraperitoneal, intravenous(systemic), subcutaneous, transdermal, oral, rectal, inhalation,topical, and intranasal.
 11. The method of claim 10 wherein theadministration route is subcutaneous.
 12. The method of claim 7 whereinthe cyclodextrin or derivative of a cyclodextrin is administered at adose ranging from about 2-20 mg/kg/day to about 4,000-20,000 mg/kg/week.13. The method of claim 12 wherein the cyclodextrin or derivative of acyclodextrin is administered at least one time per week up to aboutthree times per day.
 14. The method of claim 7 wherein the compound isadministered as a composition consisting essentially of at least onecyclodextrin or derivative of at least one cyclodextrin and at least onepharmaceutically acceptable excipient or vehicle.
 15. The method ofclaim 7 wherein the compound is administered as a composition comprisinga cyclodextrin or derivative of a cyclodextrin, a second activeingredient which is not a cyclodextrin or derivative of a cyclodextrin,and an excipient or vehicle.
 16. The method of claim 15, wherein thesecond active ingredient is selected from an antidiabetic agent, acholesterol biosynthesis inhibitor, a cholesterol absorption inhibitor,a bile acid sequestrant, niacin or niacin derivative, a fibrate, acholesteryl ester transferase protein, and an acetyl-coenzyme Aacetyltransferase inhibitor or a biologic.
 17. The method of claim 15wherein the second active ingredient is selected from animmunosuppressive agent, insulin, sulphonylurea, gliptin, metformin,thiazolidinedione, insulin sensitizer, incretin analogue, DPP4inhibitor, VEG-interfering agent, growth factor, antinflammatory,vitamin D derivative, RAS system blocker, and aldosterone blocker.
 18. Amethod for treating, inhibiting, preventing, or ameliorating acondition, or a symptom or secondary condition caused by accumulation ofcholesterol in a cell or plasma membrane of a cell, said methodcomprising administering to a patient suffering from the condition, orsymptom or secondary condition, an effective amount of a compound whichreduces cellular accumulation of cholesterol by affecting a cellularinflux mechanism or a cellular efflux mechanism relating to cholesterolaccumulation.
 19. The method of claim 18 wherein cellular accumulationof cholesterol occurs in diabetes (type 1), diabetes (type 2),prediabetes, obesity, metabolic syndrome, diabetic nephropathy, diabetickidney disease, diabetic neuropathy, diabetic retinopathy, diabetesrelated microvascular complication, diabetes related macrovascularcomplications, atherosclerosis, peripheral vascular disease, coronaryartery diseases, congestive heart failure, cardiac hypertrophy,myocardial infarction, endothelial dysfunction and hypertension, stroke,cerebrovascular accident, myocardial infarction, heart attack,cardiovascular accidents, fatty liver, steatohepatitis, NASH, insulinresistance.
 20. The method of claim 18 wherein cellular accumulation ofcholesterol results from diabetes (type 1), diabetes (type 2),prediabetes, obesity, metabolic syndrome, diabetic nephropathy, ordiabetic kidney disease.
 21. The method of claim 18 wherein the compoundinhibits a cellular influx mechanism or increases a cellular effluxmechanism relating to cholesterol accumulation.
 22. The method of claim18 wherein the cell is in a central or peripheral organ affected by orresponsible for development of diabetes.
 23. The method of calim 22wherein the organ is a pancreas.
 24. The method of claim 18 wherein thecompound modulates a sphingolipid enzyme to reduce cellular cholesterolaccumulation.
 25. The method of claim 24 wherein the compound furtherinterferes with a cellular cholesterol synthetic pathway.
 26. The methodof claim 18 wherein the compound is a cyclodextrin or a derivative of acyclodextrin.
 27. The method of claim 26 wherein the cyclodextrin ishydroxypropyl beta cyclodextrin.
 28. The method of claim 18 wherein thecompound is administered by a route selected from intramuscular,intraperitoneal, intravenous (systemic), subcutaneous, transdermal,oral, rectal, inhalation, topical, and intranasal.
 29. The method ofclaim 26 wherein the cyclodextrin or derivative of a cyclodextrin isadministered at a dose ranging from about 2 mg/kg/day to about 20,000mg/kg/week.
 30. The method of claim 26 wherein the cyclodextrin orderivative of a cyclodextrin is administered at least one times per weekup to about three times per day.
 31. The method of claim 18 wherein thecompound is administered as a composition consisting essentially of atleast one cyclodextrin or derivative of at least one cyclodextrin and atleast one pharmaceutically acceptable excipient or vehicle.
 32. Themethod of claim 18 wherein the compound is administered as a compositioncomprising a cyclodextrin or derivative of a cyclodextrin, a secondactive ingredient which is not a cyclodextrin or derivative of acyclodextrin, and an excipient or vehicle.
 33. The method of claim 32,wherein the second active ingredient is selected from an antidiabeticagent, a cholesterol biosynthesis inhibitor, a cholesterol absorptioninhibitor, a bile acid sequestrant, niacin or niacin derivative, afibrate, a cholesteryl ester transferase protein, and an acetyl-coenzymeA acetyltransferase inhibitor or a biologic.
 34. The method of claim 32wherein the second active ingredient is selected from animmunosuppressive agent, insulin, sulphonylurea, gliptin, metformin,thiazolidinedione, insulin sensitizer, incretin analogue, DPP4inhibitor, VEG-interfering agent, growth factor, antinflammatory,vitamin D derivative, RAS system blocker, and aldosterone blocker.
 35. Acomposition for treating, inhibiting, preventing, or ameliorating asymptom or related condition caused by, cellular accumulation ofcholesterol said composition comprising an effective amount of acyclodextrin or derivative of cyclodextrin, and a pharmaceuticallyacceptable excipient or vehicle.
 36. The composition of claim 35,wherein the composition consists essentially of one or more cyclodextrinor derivative of cyclodextrin as an active pharmaceutical ingredient inthe composition.
 37. The composition of claim 35, wherein thecomposition further comprises a second active pharmaceutical ingredientwhich is not a cyclodextrin or a derivative of a cyclodextrin.
 38. Thecomposition of claim 35, wherein the second active ingredient isselected from an antidiabetic agent, a cholesterol biosynthesisinhibitor, a cholesterol absorption inhibitor, a bile acid sequestrant,niacin or niacin derivative, a fibrate, a cholesteryl ester transferaseprotein, and an acetyl-coenzyme A acetyltransferase inhibitor or abiologic.
 39. The composition of claim 35 wherein the second activeingredient is selected from an immunosuppressive agent, insulin,sulphonylurea, gliptin, metformin, thiazolidinedione, insulinsensitizer, incretin analogue, DPP4 inhibitor, VEG-interfering agent,growth factor, antinflammatory, vitamin D derivative, RAS systemblocker, and aldosterone blocker.
 40. An article of manufacturecomprising a composition in a container, said composition comprising atleast one cyclodextrin or a derivative of a cyclodextrin, wherein thecontainer is packaged with written instruction for use of thecomposition for treatment of a condition caused by, or symptom resultingfrom, cellular accumulation of lipid.
 41. The article of manufacture ofclaim 40, wherein the condition is selected from diabetes (type 1),diabetes (type 2), prediabetes, obesity, metabolic syndrome, diabeticnephropathy, diabetic kidney disease, diabetic neuropathy, diabeticretinopathy, diabetes related microvascular complication, diabetesrelated macrovascular complications, atherosclerosis, peripheralvascular disease, coronary artery diseases, congestive heart failure,cardiac hypertrophy, myocardial infarction, endothelial dysfunction andhypertension, stroke, cerebrovascular accident, myocardial infarction,heart attack, cardiovascular accidents, fatty liver, steatohepatitis,NASH, and insulin resistance.
 42. The article of manufacture of claim 40wherein the condition is selected from obesity, metabolic syndrome,pre-diabetes, diabetes, diabetic kidney disease or conditions orsymptoms relating thereto.
 43. The article of manufacture of claim 35wherein the cellular lipid is cholesterol.